In Vivo Expression from the RpoS-Dependent P1 Promoter of the Osmotically Regulated proU Operon in Escherichia coli and Salmonella enterica Serovar Typhimurium: Activation by rho and hns Mutations and by Cold Stress

Abstract

Unlike the 70 -controlled P2 promoter for the osmotically regulated proU operon of Escherichia coli and Salmonella enterica serovar Typhimurium, the s -controlled P1 promoter situated further upstream appears not to contribute to expression of the proU structural genes under ordinary growth conditions. For S. enterica proU P1, there is evidence that promoter crypticity is the result of a transcription attenuation phenomenon which is relieved by the deletion of a 22-base C-rich segment in the transcript. In this study, we have sought to identify growth conditions and trans-acting mutations which activate in vivo expression from proU P1. The cryptic S. enterica proU P1 promoter was activated, individually and additively, in a rho mutant (which is defective in the transcription termination factor Rho) as well as by growth at 10°C. The E. coli proU P1 promoter was also cryptic in constructs that carried 1.2 kb of downstream proU sequence, and in these cases activation of in vivo expression was achieved either by a rho mutation during growth at 10°C or by an hns null mutation (affecting the nucleoid protein H-NS) at 30°C. The rho mutation had no effect at either 10 or 30°C on in vivo expression from two other s -controlled promoters tested, those for osmY and csiD. In cells lacking the RNA-binding regulator protein Hfq, induction of E. coli proU P1 at 10°C and by hns mutation at 30°C was still observed, although the hfq mutation was associated with a reduction in the absolute levels of P1 expression. Our results suggest that expression from proU P1 is modulated both by nucleoid structure and by Rhomediated transcription attenuation and that this promoter may be physiologically important for proU operon expression during low-temperature growth.The ProU transporter in Escherichia coli and Salmonella enterica serovar Typhimurium is a binding-protein-dependent transport system that mediates the cytoplasmic accumulation of compatible solutes such as glycine betaine, L-proline, and related compounds during growth of cells in media of elevated osmolarity (9, 10). The subunit polypeptides of the transporter are encoded by three genes, proV, proW, and proX, which together constitute (in that order) the proU operon (16).Transcription of proU in both E. coli and S. enterica is activated several-hundredfold in cultures grown in high-osmolarity media, but the mechanism of osmotic induction of the operon is not fully understood (reviewed in references 10, 19, and 29). Two cis regulatory elements that have been identified (see Fig. 1) include a 70 -driven promoter whose transcription start site is situated 60 bases upstream of proV (16, 24, 28, 54) and a negative regulatory element (NRE) approximately 500 bp long, which is situated downstream of the promoter (overlapping the proV coding region) and whose deletion results in partial derepression of proU at low osmolarity (11,24,37,38). Mutations in hns, the gene encoding an abundant nucleoid protein, H-NS, also result in partial derepression of proU expression (for a review of H-NS...

The Degree of Oligomerization of the H-NS Nucleoid Structuring Protein Is Related to Specific Binding to DNA

“…The documented flexibility of the two-domain wild-type structure could favor such a rearrangement. Furthermore, it is reported that, in vivo at proU, a temperature increase leads also to an activation of the P1 promoter (32). Therefore, changes in the strength of protein-protein contacts as well as DNA conformational changes (postulated by Falconi et al (9)) could contribute to the sharp regulation exerted by temperature on prokaryotic promoters controlled by H-NS.…”

An N-Terminally Truncated RpoS (σ S ) Protein in Escherichia coli Is Active In Vivo and Exhibits Normal Environmental Regulation Even in the Absence of rpoS Transcriptional and Translational Control Signals

“…2), indicating that it is a hot-spot site for transpositions involving IS10 and its derivative composite transposons. Furthermore, data from an earlier study (27) indicated that the rho mutation does not restore proU P1-lac expression in strains carrying the rpoS359::Tn10 mutation of RH90.…”

“…MacConkey lactose agar was obtained from Difco. Cultures for ␤-galactosidase assays were incubated with shaking (i) until mid-exponential phase (A 600 of 0.4 to 0.8) or until 1 h after entry into stationary phase; (ii) at 30 or 10°C; and (iii) in LB medium or low-osmolarity K-tryptone medium (27), the latter supplemented when necessary with NaCl to 0.3 M. Unless otherwise indicated, Ara induction experiments (11) were performed by supplementation of the growth medium with 0.2% Ara (or 0.2% Glu, as a control). Concentrations of antibiotics used were as described earlier (20).…”

“…It was shown earlier that the P1 promoter of the E. coli proU operon is S regulated both in vivo and in vitro (20,(27)(28)(29); its in vivo activity (for example, in a wild-type strain such as GJ862) ( Table 2) can be assayed by using plasmid pHYD275, which is a very-low-copy-number replicon that carries P1 upstream of the lacZ reporter gene (4). The starting point for this study were the unexpected observations ( Table 2) that lac expression from pHYD275 was abolished in one of our laboratory strains, GJ829, but that it was restored in an isogenic derivative, GJ830, that was defective in the rho gene (encoding the transcription termination factor Rho; for a review, see reference 31).…”

“…The starting point for this study were the unexpected observations ( Table 2) that lac expression from pHYD275 was abolished in one of our laboratory strains, GJ829, but that it was restored in an isogenic derivative, GJ830, that was defective in the rho gene (encoding the transcription termination factor Rho; for a review, see reference 31). The rho mutation had little effect in a wild-type strain ( Table 2, compare values for GJ862 and GJ863; see also reference [27]). An rpoS::Tn10 derivative of GJ830 was also negative for pHYD275-driven lac expression (data not shown), indicating that the rho mutation acted specifically and in a S -dependent manner to restore proU P1-lac expression in GJ829.…”